Methods for heat-assisted enzyme digestion

ABSTRACT

The present disclosure relates to a method of digesting a sample comprising protein, the method comprising: adding the sample to an apparatus containing a buffer and a solid support surface comprising a surface coating, wherein the surface coating immobilizes enzyme while reducing undesired interactions between the sample and the solid support surface; immobilizing enzymes on the surface coating for digestion of the protein of the sample; and heating the sample to complete a heat-assisted digestion of the protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority and benefit to U.S. Provisional PatentApplication No. 63/022,056 filed on May 8, 2020, entitled “Methods forHeat-Assisted Enzyme Digestion.” The content of which is incorporatedherein by reference in its entirety.

FIELD OF THE TECHNOLOGY

The present disclosure relates to methods for heat-assisted biomoleculesample processing, e.g., digestion and affinity ligand purification.More specifically, the present disclosure relates to the use of acoating, such as a hetero-functional coating, for conjugatingbiomolecules on a solid support surface in combination with heat forsample processing (e.g., clean up, sample release) for bioanalysis.

BACKGROUND

Biomolecules are complex molecules, which require complex workflows foranalysis. These complex workflows involve numerous steps includingvarious sample preparation steps, such as, sample clean up and proteindigestion. Obtaining substantially complete digestion while noteffecting sample quality (e.g., not introducing byproducts which requireadditional cleanup) is challenging. For example, some enzymes used indigestion when used in solution can become unstable, leading tobyproducts. Conducting the digestion at elevated temperatures canimprove the throughput of workflow. However, enzymes are most ofteneffective within a specific temperature range due to denaturation orother conformation change induced by the heat. But at the specifictemperature range of the enzyme, autolysis of the protein can bedramatically increased. Additionally, the hardware for the source ofheat can be difficult to benchmark and assess for reproducibility.

SUMMARY

Biomolecules are complex molecules, which require complex workflows foranalysis. These complex workflows involve numerous steps includingvarious sample preparation steps, such as, sample clean up and proteindigestion. Obtaining substantially complete digestion while noteffecting sample quality (e.g., not introducing byproducts which requireadditional cleanup) is challenging. For example, some enzymes used indigestion when used in solution can become unstable, leading tobyproducts.

However, enzymes are most often effective within a specific temperaturerange due to denaturation or other conformation change induced by theheat. But at the specific temperature range of the enzyme, autolysis ofthe protein can be dramatically increased. Additionally, the hardwarefor the source of heat can be difficult to benchmark and assess forreproducibility.

Immobilization of these enzyme helps to stabilize the enzymes. However,the conventional techniques used to immobilize enzymes lead to secondaryinteractions with the surface of the support, impacting digestionefficiency and sample recovery.

With the immobilized enzymes, the digestion temperature can beincreased. And the drawbacks associated with elevated temperatures canbe reduced. The result is increased workflow without sacrificing thequality of the digestion.

In general, the present disclosure is directed to a fast digestionmethod assisted by heat to efficiently complete digestion in minuteswhile providing high-fidelity peptide profiles, which is reliable andsuitable for biology research or protein therapeutics characterizationin a regulated environment. High-fidelity is defined as the digestionworkflow results that provide >95% sequence coverage, <10%miss-cleavage, comparable modification (approximately <5% and in someexamples less than 1% of heat-induced modification after digestionconducted at 25-100° C.) for therapeutic proteins in comparison toconventional in-solution digestion workflow.

The high-fidelity digestion workflow is used for peptide mapping,disulfide bond mapping, middle-up, middle-down, bottom-up proteomics,protein identification, protein quantification, bioanalysis, otherdigestion-related applications, or other applications (e.g.,applications using affinity ligands). The method is comprised ofheat-stable enzyme(s), digestion buffer (or other types of buffers),heat source, and steps to get sample ready for downstream analysis(fluorescence, UV, LC-MS detection) either through clean-up or reactionquenching.

The present disclosure provides methods for conducting fast enzymedigestion with the assistance of heat. The fast digestion method can beused for protein/peptide analysis. In general, the method includes oneor more of the following: heat-stable enzyme(s), digestion buffer, heatsource, and steps to set sample ready for downstream analysis(fluorescence, UV, LC-MS detection) either through clean-up or reactionquenching. For example, some of the methods include the followingscomponents: the immobilized enzyme on a solid support with superior heatstability and a hydrophilic characteristic that minimizes non-specificbinding; the digestion buffer that supports the kinetics of enzyme (suchas trypsin) activity at elevated temperature while also minimizing theheat-induced modification on peptides; the apparatus (e.g., vials,plates or columns) ensures maximum heat transfer efficiency with minimalnon-specific binding.

The present disclosure provides a method of processing a samplecomprising protein, the method includes: adding the sample to anapparatus containing a buffer and a solid support surface comprising asurface coating, wherein the surface coating immobilizes enzymes andaffinity ligands while reducing undesired interactions between thesample and the solid support surface; immobilizing enzymes on thesurface coating for digesting the protein in the sample; immobilizingaffinity ligands on the surface coating for target capturing the proteinin the sample; digesting the protein in the sample with the immobilizedenzymes on the surface coating; target capturing a portion of the samplewith the immobilized affinity ligands on the surface coating; andheating the sample to activate digestion of the protein. In someembodiments, the step of target capturing a portion of the sample occursbefore digestion of the protein. In certain embodiments, the step oftarget capturing a portion of the sample occurs during digestion. Insome embodiments, the step of target capturing a portion of the sampleoccurs after digestion. And in some embodiments, the step of targetcapturing a portion of the sample occurs before, during, and/or afterdigestion of the protein. In some embodiments, the buffer is selectedfrom the group consisting of Tris, BIS-Tris, 2-ethanesulfonic acid(MES), HEPES, triethanolamine, and trimethylamine. The buffer cancontain divalent ions such as CaCl₂. The buffer in-solution can containpolyols selected from a group consisting of, and not limited to,glycerol, xylitol, propylene glycol, butanediol or erythritol. In someembodiments, the buffer in-solution includes xylitol and CaCl₂. In someembodiments, methionine is added to the buffer in-solution. In someembodiments, the buffer in-solution further includes one or moreadditives, such as for example, xylitol, methionine, and/or CaCl₂.

BRIEF DESCRIPTION OF THE DRAWINGS

The technology will be more fully understood from the following detaileddescription taken in conjunction with the accompanying drawings, inwhich:

FIG. 1 is a graph of thermograms in accordance the present disclosure.

FIG. 2 is a graph of thermograms in accordance the present disclosure.

FIG. 3A is a graph illustrating the effect on hydrophobic peptiderecovery of adding arginine to a digestion buffer. FIG. 3B is a graphillustrating the effect on digestion efficiency of adding arginine tothe digestion buffer. FIG. 3C is a graph illustrating the effect onhydrophobic peptide recovery of adding dimethyl-arginine to a digestionbuffer. FIG. 3D is a graph illustrating the effect on digestionefficiency of adding dimethyl-arginine to the digestion buffer.

FIG. 4 is a graph of a recovery of a mixture of hydrophobic peptidesafter incubation with immobilized enzymes in accordance with the presentdisclosure.

FIG. 5 is a graph of released trypsin after incubated in digestionbuffer at 70° C.

FIGS. 6A, 6B, and 6C are graphs displaying LC-MS chromatograms generatedafter NIST mAb digested under varying conditions.

FIG. 7 is a graph displaying miss-cleavage% and sequence coverage %comparing examples in accordance with the present disclosure.

FIGS. 8A, 8B, and 8C are graphs comparing digestion examples atdifferent temperatures.

FIGS. 9A and 9B are graphs comparing the effect of Ca²⁺ concentration ondigestion efficiency and non-specific binding.

FIGS. 10A and 10B are graphs comparing the effect of pH on digestionefficiency and deamidation %.

FIGS. 11A and 11B are graphs comparing the effect of Tris concentrationon digestion efficiency and non-specific binding.

FIGS. 12A and 12B are graphs comparing the effect of polyols ondigestion efficiency and non-specific binding. FIG. 12C is a graphdisplaying the comparison of digestion results using glycerol andxylitol.

FIG. 13 is a graph displaying the effect of methionine on preventingartificial oxidation.

FIG. 14 is a graph displaying the effect of acetonitrile on reducingartificial deamidation.

FIG. 15 is a graph displaying digestion efficiency comparison amongexamples.

FIG. 16 displays a typical reaction set-up in a 200 μL PCR tube.

FIG. 17 displays dimensions of PCR tubes from the tubes used in FIG. 15.

FIG. 18 is a flowchart of an example of trypsin digestion currentworkflow.

DETAILED DESCRIPTION

Conducting protein digestion at elevated temperature has beenproblematic because of incompleteness of the digestion, nonspecificbinding to the resin, and heat-induced modifications on peptidescompared to traditional in solution digestion that often carried out at37° C. Through engineering an immobilized enzyme with a specific surfacechemistry, the present disclosure facilitates a workflow that generatesa highly similar profile as in-solution digestion but only requiresminutes of digestion. The digestion buffer has been tailored to increasepeptide recovery and enzyme activity.

Of note, the digestion completeness of the present disclosure is 90-95%,much higher compared to the leading competitor product SMART Digest™(available from Thermo Fisher Scientific, Waltham, Mass.) which is only65%-70%. The non-specific binding effect of peptides is reduced by10-30% using the technology of the present disclosure compared to SmartDigest™. The present disclosure is also amenable to reducing/alkylatingreagents while Smart Digest™ is not compatible.

Enzyme digestion of proteins is widely adopted in the biochemistryresearch community, where the resulted peptides could inform on theamino acid modification or the abundance of the protein in a relevantphysiological environment. With the emergence of antibody therapeuticsin biopharmaceutical industry, this technique has proved its value inthe bioprocess and quality control evaluations to support theidentification and monitoring of critical quality attributes thatreflect the purity and safety profiles of a particular therapeutic.However, the digestion of a large protein, e.g. an antibody with 150 kDamolecular weight, takes hours to complete and oftentimes requiresoptimization on many experimental parameters including incubation time,temperature, and protein to enzyme ratios. This lengthy process hasrestricted the throughput where timely decisions often need to be madeeither in the discovery phase or in the manufacturing process. Anothercaveat of the process is the lengthy digestion process could induceartificial modification on the proteins or peptides, adding extra workin defining those modifications. In the case of trypsin digestion,autolysis along with time could result in unfavorable interference fordownstream analysis.

Attempts to increase the throughput of the workflow have inspired somerecent advances in automating the workflow. Liquid handlers now cansimultaneously process 96 or even 384 samples at one time, however thetime for digestion itself still requires hours. On the other hand, themost common downstream peptide analysis requires a 60-120 minutegradient of liquid chromatography to achieve sufficient separation, sothere is a maximum of 24 samples that can be processed a day. Excessnumber of samples queued in line may suffer from potential loss ofpeptides due to stability or adsorption issues. This would in turnaffect the consistency and accuracy of the platform assay.

Attempts to conduct digestion at elevated temperature also have beeninvestigated. The rationale behind is that enzyme reaction, just likeany other chemical reaction, follows the rule of the Arrhenius equation(Equation 1):

$\begin{matrix}{k = {Ae^{\frac{{- E}a}{RT}}}} & (1)\end{matrix}$

Where k is the kinetic rate constant for the reaction, A is theArrhenius constant, G is the standard free energy of activation (kJ M⁻¹)which depends on entropy and enthalpy factors, R is the gas law constantand T is the absolute temperature. Typical standard free energies ofactivation (15-70 kJ M⁻¹) give rise to increases in rate by factorsbetween 1.2 and 2.5 for every 10° C. rise in temperature. But usuallythe enzyme would be most effective within a preferred temperature rangedue to denaturation or other conformation change induced by heat. Usingsequencing grade trypsin as an example, the preferred temperature is50-55° C. for a 1-hour digestion experiment on casein. However, at suchtemperature the autolysis of trypsin has dramatically increased alongwith time which resulted in a 60% activity loss within 2 hours.

The heat source can be a heat source that provides uniformity (uniformheat transfer) and even heat distribution. In the example of immobilizedenzyme, agitation of a dispersive device or flow though mode interactionwith protein needs to be ensured to achieve a homogenous or evencontact. The ramp time that the heat source can provide is also a factorwhen selecting the heat source. A short ramp time is advantageous. Inthe dispersive immobilized enzyme format, 5 minutes or less time is usedto reach to the determined digestion temperature for a 10-minutedigestion. In one example of immobilized trypsin, 5 minutes or less toreach to 75° C. and maintain the temperature at 75° C. is preferred.

In some examples, heat source can be anything that supports consistentheating at 50-100° C. for 5 minutes or longer. Some examples include anoven, incubator, rocker, or thermomixer. In some examples, to achieveconsistent and best result Eppendorf ThermoMixer C (based on Peltiertechnology) can be used, preferably with the option of a heated lid. Theheated lid can be helpful for digestions that take a long time (e.g.,over 30 minutes).

Efficient heat transfer between the consumable and heat source is also afactor when selecting the heat source. In one example of dispersiveimmobilized trypsin, the thickness of consumables used correlated withtheir digestion performance. The preferred temperature depends on theenzyme or combination of enzymes selected. In the example of immobilizedtrypsin, a T_(m) and onset temperature (T_(onset)) can help determine atwhat temperature is most appropriate for a heated digestion. Thepreferred temperature for trypsin is between T_(onset) to T_(m).

Microwave-assisted and infrared-assisted enzyme digestion appeared toimprove the kinetics of digestion by shortening the time to as little as5 min, however these studies were mostly helpful for small to mediumsize proteins (below 100 kDa) and the quality of the digested productshave not been rigorously evaluated. Moreover, hardware like microwave asthe source of heat is simply difficult to benchmark and assessed forreproducibility.

Immobilized enzyme in dispersive format, or in column/cartridge formatare also commercially available. The advantage of immobilization is toimprove the heat-stability by restraining the denaturation effect ofheat so that digestion could be completed at a higher temperature withina shortened period of time. However, by far the reported workflows couldonly achieve limited digestion efficiency (the percentage of all trypticpeptides vs. total peptides detected) varying from 30% to 80% amongproteins compared to in-solution digestion. Most protocols associatedwith immobilized enzyme advocated the elimination of pretreatment(denaturation, reduction and alkylation), claiming sufficientdenaturation achieved by heat. However, this only applies to proteinsare small or prone to heat denaturation, since the incomplete digestionfor larger proteins or proteins with multiple disulfide bonds will notbe efficiently denatured by heat. Moreover, the extent of heat-inducedmodification and the reproducibility of these innovations that haverarely been evaluated rigorously and extensively.

FIG. 18 is a flow chart illustrating the peptide mapping workflow 700.In some examples, peptide mapping workflow 700 includes four parts. Apart one 702 includes a sample with an analyte of interest, such as aprotein, is unfolded. A part two 704 includes desalting the sample,which includes the unfolded analyte of interest. A part three 706includes digesting the analyte of interest of the sample. Here, thedevice used in digesting the analyte of interest includes ahetero-functional coating of the present disclosure. After the analyteof interest is digested, a part four 708 includes collecting the samplewith digested analyte of interest.

In some examples, part one 702 and part two 704 can be dependent on theanalyte of interest. For example, part one 702 and part two 704 can beconsidered pre-treatment steps and may not be required based on theanalyte of interest, such as a protein.

A desired fast digestion method assisted by heat would efficientlycomplete digestion in minutes while providing high-fidelity peptideprofiles, which would be reliable and suitable for either biologyresearch or protein therapeutics characterization in a regulatedenvironment. High fidelity in this context is interpreted as the resultsof the digestion workflow provide >95% sequence coverage, <10%miss-cleavage, comparable modification% (usually <5%) for therapeuticproteins in comparison to conventional in solution digestion workflow.This disclosure provides the methods for conducting fast enzymedigestion with the assistance of heat. The methods comprise essentiallythe following components: the immobilized enzyme on a solid support withsuperior heat stability and a hydrophilic characteristic that minimizesnonspecific binding; the digestion buffer that supports the kinetics oftrypsin activity at elevated temperature while also minimizes theheat-induced modification on peptides; the apparatus (vials, plates orcolumns) that ensures the maximum heat transfer efficiency with minimalnonspecific binding.

The present disclosure provides the enhanced methods for conducting fastenzyme digestion with the assistance of heat. The methods compriseessentially the following components: The immobilized enzyme on a solidsupport with superior heat stability and a hydrophilic characteristicthat minimizes nonspecific binding; the digestion buffer that supportsthe kinetics of trypsin activity at elevated temperature while alsominimizes the heat-induced modification on peptides; The apparatus(vials, plates or columns) that ensures the maximum heat transferefficiency with minimal nonspecific binding. The complete digestionworkflow starts with desaturating proteins in 8M guanidine or 6M urea.After reduction and alkylation, the proteins are desalted and subject todigestion. The digestion mixture is composed of immobilized enzyme,proteins in digestion buffer at beneficial concentrations. Relevantfactors for the digestion buffer can be temperature, pH, metalconcentration, and additives.

The buffer of the present disclosure can include a buffering agent tocontrol pH, protein sample dispersant and metal ions. The metal ions canbe divalent metal ions, preferably Ca²⁺. The buffering agent can includeTris, BIS-Tris, MES, HEPES, Triethanolamine, and trimethylamine. Theprotein sample dispersant can be chosen from a group of polyols such asglycerol, xylitol, propylene glycol, butanediol or erythritol. The metalions and protein sample dispersant can be additives in the buffer. Forexample, CaCl₂, methionine, xylitol, and/or glycerol can be additives inthe buffer.

After digestion the immobilized enzyme are removed either throughcentrifugation or filtration. Peptides are recovered as supernatants andsubmitted to downstream analysis, either LC-UV or LC-MS.

An immobilized enzyme (or in some examples an affinity ligand) on thecoating, as described in more detail in Appendix A, provides heatstability. As for digestion, chemical immobilization methods through acovalent interaction approach stand out since this would ensure theleast enzyme leakage, and more importantly, better heat stability. Manyof these immobilized enzymes are provided in a column format which oftenrequires multi-dimensional LC systems for operation. The workingtemperature varies from 37° C. to 60° C. and there is a lack ofinformation on their lifetime and reproducibility. One notablecommercially available product in a dispersive format is Smart Digest™which has adopted a working temperature at 70° C.

FIG. 1 is a graph of thermograms in accordance with aspects of thepresent disclosure. Specifically, FIG. 1 displays NanoDSC thermograms offree trypsin, Smart Digest™ trypsin, and one preferred immobilizedenzyme (e.g., trypsin) prototype (e.g., silica-based solid support withcoating in accordance with the present technology), whereT_(m)—temperature where half of the protein is unfolded andT_(onset)—temperature where protein starts to unfold. For example, theprototype (i.e., silica-based solid support with coating) withimmobilized trypsin showed even better thermostability compared to SmartDigest™ (FIG. 1).

FIG. 2 is a graph of thermograms in accordance with aspects of thepresent disclosure. FIG. 2 displays NanoDSC thermograms of immobilizedenzyme prototypes with different modification. The unmodified tryspinhas a T_(m) around 65° C. The point modified trypsin (butyl acrylate,s-methylisothiurea and phenyl glyoxal have slightly improved T_(m) for1-2° C., while crosslinker modified trypsin (glycerol 1,3 diglycerolatediacrylate and triethlyleneglycoldiacrylate) has significantly improvedT_(m) for 5-7° C. To further increase the heat stability of trypsin,crosslinkers that nonspecifically react with amines could restrain thestructural change under heat (FIG. 2). Similarly, modifiers thatcovalently bound with a certain amino acid that are not at the activesites also could create steric hindrance during heat denaturation (FIG.2).

The immobilized enzymes on the support impart enhanced surfacecharacteristics, such as nonspecific binding, conjugation chemistry, anddigestion efficiency. The solid support can be composed of a hydrophilicsurface with porous structure that could minimize non-specific binding,which in turn would ensure the digestion efficiency and the recovery ofpeptides. Important physical parameters include the particle size andpore size of the solid support, which affect the diffusion of proteinsinto the pores and how much access they have to the enzyme. For aselected material, hydrophilic modification is optimized so that thenonspecific binding could be tuned to a preferred level. FIG. 4 is agraph of a recovery of a mixture of hydrophobic peptides afterincubation with immobilized enzymes in accordance with the presentdisclosure. FIG. 4 demonstrate the recovery % of hydrophobic peptidesafter mixed with selected immobilized support for only 5 minutes.

In some examples, the solid support is porous and the pore size rangesfrom about 50 to about 5000 Å. The pore size can affect the diffusion ofthe protein into the pores and how much access the protein has to theenzyme.

The solid support surface can be a plurality of particles, each particlehaving a particle size ranging from about 1 to about 200 microns. Theparticle size can affect the diffusion of the protein and how muchaccess the protein has to the enzyme. The solid support is the materialfor immobilizing the enzymes. In some examples, the consumable used forcontaining the reaction components includes a multi-well plate, a vial,or a column. The consumable be coated to alleviate non-specific binding.

Another consideration on immobilization is how stable the enzyme isconjugated on the support. The released enzyme usually has significantlyreduced thermal stability at elevated temperature and with less enzymeimmobilized on the support, the digestion efficiency would decreasealong with time. Worse still, in the case of trypsin, it can contributeto significant noises to downstream analysis with the autolysis productsif UV analysis is employed for downstream analysis. FIG. 5 is graph ofreleased trypsin after incubated in digestion buffer at 70° C. Apreferred product has minimal leakage of the enzyme during the digestion(FIG. 5).

In some examples, immobilized trypsin does not show autolysis at lowtemperature (25-45° C.), very low autolysis (less than 1%) attemperatures above 65° C. at pH greater than 7, and no autolysis atlower pH.

The immobilized enzyme can include one enzyme or combination of enzymes(e.g., trypsin, chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain,pepsin, elastase, IdeS, pronase and PNGase) could be immobilized onparticle, chip, surface that could sustain certain elevated temperaturewithin 25-110° C. and do not negatively affect enzyme activity. In someexamples, elevated temperature range may be further narrowed for heatingto occur at an elevated temperature ranging above 45° C. preferably 65°C. to 75° C. and not greater than 85° C. Enzyme can possess heatstability through the immobilization process. In the example of trypsin,melting temperature (Tm) can be by increased 30-50° C. afterimmobilization. Modification of trypsin through point modification andcrosslinking and conjugating small polymers or ligands can furtherincrease Tm. In some examples, the solid support with the immobilizedenzyme has less than 30% non-specific binding for all protein/peptidespresent during digestion, and the immobilized enzyme can be stable withleaching less than 20% during the digestion procedure.

In some examples, the modified enzyme is in-solution. The enzyme caninclude one enzyme or a combination of enzymes (e.g., trypsin,chymotrypsin, Lys-C, Glu-C, Arg-C, Asp N, papain, pepsin, elastase,IdeS, pronase and PNGase).

The digestion efficiency of an immobilized enzyme is a combined resultof surface characteristics. Using NIST mAb as a model protein, themiss-cleavage % could reflect the digestion efficiency by weighing theratio of tryptic peptides (with no miss-cleavage) in the total peptidesdetected after digestion. Reported sequence coverage for using SmartDigest™ under recommended workflow could only reach 50-76% for IgG1.FIGS. 6A, 6B, and 6C are graphs displaying LC-MS chromatograms generatedafter NIST mAb digested under varying conditions. Specifically, FIGS.6A, 6B, and 6C are graphs displaying LC-MS chromatograms generated afterNIST mAb digested under (FIG. 6A) two hour in solution digestion; (FIG.6B) 10-minute Smart Digest™ digestion; (FIG. 6C) 10-minute prototypedigestion. FIG. 7 is a graph displaying miss-cleavage % and sequencecoverage % comparing examples in accordance with the present disclosure.Specifically, FIG. 7 displays miss-cleavage% and sequence coverage %compared among prototypes with increasing levels of hydrophilicmodification and Smart Digest™. A desired prototype with improved heatstability should be capable to provide robust digestion (miss cleavage%<10%, sequence coverage>90%) under optimized condition (FIGS. 6 & 7).By tuning the hydrophilic modification on the surface, the digestionefficiency could find a “sweet spot” (FIG. 7) with a balance ofdigestion efficiency and mass recovery of peptides.

Before digestion, the present disclosure can include pretreatment.Before protein digestion, there can be a pretreatment step. In someexamples, proteins that can be easily denatured by heat and areintroduced during digestion do not require pretreatment. For proteinsthat need pretreatment, denaturation followed with reduction andalkylation are common steps to fully unfold the protein.

The digestion conditions play important role for digesting animmobilized enzyme at elevated temperatures. Digestion parameters likepH, temperature and digestion time that work best with free enzyme insolution will exhibit distinctive features in a heated environment basedon the selection of immobilization support. Additives can help cope withimproved kinetics, heat-induced modification on peptides, adsorption ofpeptides onto the immobilized support and aggregation of slurry orproteins. With heat-assisted denaturation, complete digestion could beachieved without pre-treatment including denaturation with guanidine orurea, reduction and alkylation, without identifying the extent ofmiss-cleavage. In the present disclosure, complete digestion as theresult of miss-cleavage % and is no bigger than 10%, which means theprotein have been completely digested in a way that more than 90% of theresulted peptides have no miss-cleaved sites. This is a stringentcriterion and also the quality that differentiates the presentdisclosure and the utilization of the present disclosure from others.

In the present disclosure, pre-treatment is optional depending on theanalyte of interest (e.g., depending on the protein). Desalting toremove pre-treatment is optional because the resin is compatible.Pre-treatment of samples (including denaturation, reduction andalkylation) can be conducted before digestion in pursuit ofhigh-fidelity peptide profiles. While the whole process, includingpre-treatment, is no longer than 2 hours with digestion included.

After immobilization the enzyme exhibits improved thermal stability, anda preferred temperature needs to be determined for the selected enzymesince they may exhibit distinctive correlation or no correlation betweenT_(m) and temperature for maximum activity. Empirically in the case oftrypsin, it's preferable to conduct digestion at 70-75° C. for 10 minwhich is close to its T_(m). FIGS. 8A, 8B, and 8C are graphs comparingdigestion examples at different temperatures. Specifically, FIGS. 8A,8B, and 8C display LC-MS chromatograms comparing Smart Digest™ andimmobilized trypsin after digesting NIST mAb for 10 minutes at (FIG. 8A)60° C.; (FIG. 8B) 70° C.; (FIG. 8C) 80° C.

Lower than this temperature significant incomplete digestion wouldappear (FIGS. 8A-8C). Without wishing to be limited by theory, thedigestion efficiency would reach a higher value at temperatures higherthan 70° C., and a shortened digestion time is expected to achievesimilar digestion completeness. But that also may require a slightlylonger equilibration time for the reaction mixture which could give riseto heat-induced degradation or adsorption, or other unfavorablemodifications.

In some examples, buffer composition includes calcium ions (Ca²⁺). Theactivity of trypsin in its free form could be improved by 1-10 mM Ca²⁺in solution. With the effect of ionic strength to trypsin, while notbeing bound by theory, there may be a “sweet-spot” concentrationregardless of the inorganic salts. Once immobilized, the enzymaticreaction could be carried out at a much higher temperature. For example,with trypsin, the enzymatic reaction could be carried out at 70° C. Insome examples with significantly improved kinetics, the concentrationfor catalyst Ca²⁺ may need to be re-evaluated. FIGS. 9A and 9B aregraphs displaying the effect of Ca²⁺ concentration (1-50 mM) ondigestion efficiency and non-specific binding. In one example, thedigestion efficiency has increased significantly when the concentrationof Ca²⁺ has increased from 1 mM to 50 mM (FIG. 9A). However, Ca²⁺ at 50mM also showed the most severe loss over a hydrophobic peptide (m/z 934)(FIG. 9B). A balance for enzyme activity and nonspecific binding may beachieved with 10-20 mM Ca²⁺ for most proteins, especially antibodytherapeutics whose peptide profiles are very complex.

Most enzymes exhibit pH preference. For example, trypsin in solution hasmaximized activity at pH 8.0. The buffers that are used to create suchenvironment are mostly zwitterionic compounds that vary in pKa values,which affect the buffering capacity. With elevated temperature, pKavalues usually decrease, which could induce a pH shift of the digestionbuffer. For example, the pH of 50 mM Tris solution dropped 0.6 from 25°C. to 75° C. (Table 2, shown below).

FIGS. 10A and 10B are graphs comparing effect of pH on digestionefficiency and deamidation %. In the example of FIG. 10A, the pH forimmobilized trypsin was shifted to 7.0 at 75° C. from 7.6 at 25° C.(FIG. 10A). There are situations where a lower pH is preferred. Forexample, a heated digestion is known to exert an acceleration ofdeamidation, but it could be circumvented by adjusting the pH to aslightly lower value (FIG. 10B) or simply shortening the digestion time.

In some examples, a preferred pH for the enzyme can be the sameregardless of what temperature the digestion happens. But buffers havedifferent capacity to maintain the same pH across a wide temperaturerange. In the example of immobilized trypsin, the pH for the buffer is7.6 at room temperature. At 75° C., the buffer pH has dropped to 7.0.But with the effect of heat, the digestion could occur at a faster speedso even though trypsin was not in a preferred state, the digestion stillachieved quality results of digestion in 10 minutes.

Ionic strength is another buffer composition condition. Each buffer saltat certain concentrations provides ionic strength for an enzymaticreaction to occur. Tris buffer is commonly used for digestion but theconcentration of Tris needs to be tailored for the enzyme selected fordigestion. FIGS. 11A and 11B are graphs comparing the effect of Trisconcentration on digestion efficiency and non-specific binding. In theexample of immobilized trypsin, 100 mM of Tris provides best results forbalanced digestion efficiency and enhanced peptide recovery (FIG. 11).

Once the immobilized enzyme is chosen for digestion, a thermal mixer isneeded to avoid the physical settlement of the resin to ensure an evenand efficient heat distribution during the process. However, acceleratedaggregation could occur among particles and proteins, for example,denatured proteins may aggregate much faster than in solution with morereadily hydrophobic moieties exposed under heat. Polar additives couldplay an important role to prevent unfavorable interaction betweenparticles or proteins and nurture an aqueous-like environment. In fact,small polyols including but not limited to glycerol, xylitol,erythritol, propylene glycol, and butanediol could stabilize aggregationat high temperature and salt concentration and provide high recovery %of peptides, given its hydrophilicity properties. FIGS. 12A and 12B aregraphs comparing the effect of polyols on digestion efficiency andnon-specific binding. FIG. 12C is a graph displaying the comparison ofdigestion results using glycerol and xylitol. In some examples, thehigher the hydrophilicity, the better the digestion efficiency is (FIG.12).

In the present disclosure, the enzyme activity control includedcofactor/stabilizer, ionic strength. In the example of immobilizedtrypsin, calcium concentration can be tailored to ensure the activity ofenzyme. In the example of immobilized trypsin, Tris concentration canalso be tailored according to the activity.

In the present disclosure, the non-specific binding control applies toimmobilized enzyme. In the example of immobilized trypsin, 5% glyceroland others have effect to alleviate hydrophobic peptide adsorption ontothe surface of resin. In the example of immobilized trypsin, calcium andTris concentration can be tailored for minimal nonspecific binding.

As is always a concern for process-induced modification associated withelevated temperature, there are additives that could circumvent thisproblematic effect. FIG. 13 is a graph displaying the effect ofmethionine on preventing artificial oxidation. For example, the additionof sufficient amount of oxidation scavengers, such as methionine, couldsufficiently reduce artificial oxidation (FIG. 13). In the case ofdeamidation, the addition of organic solvent, for example, 10%acetonitrile could modulate the dielectric strength and alleviatemethod-induced deamidation (FIG. 14).

A kit can include a digestion buffer to support enzymatic activity at anelevated temperature and minimize heat-induced modifications on theprotein. In some examples, less than a 1% relative conversion rate for amethionine containing peptide is to be converted to an oxidized variant.In some examples, less than a 5% relative conversion rate for anasparagine residue is to be deamidated to an isoaspartic acid oraspartic acid, preferably less than 2%, during the course of the kitprocedure. In some examples, within 10 minutes of heated digestion at pH7.5, there is no more than 1% of artificial oxidation on methionine andno more than 5% of deamidation.

Oxidation scavengers like methionine can be added to digestion buffer.In one example of immobilized trypsin, 50 mM of methionine was able toeliminate heat-induced modification compared to in-solution digestion.

Deamidation is a pH-driven process. Lowing the pH or adjusting thecomposition of buffer can prevent head-induced deamidation. In theexamples of immobilized trypsin, 10% of acetonitrile can significantlyreduce artificial deamidation.

To achieve complete digestion, time-dependent studies are needed foreach enzyme with the protein digested. In the example of immobilizedtrypsin, the digestion can be completed in 1-10 minutes.

According to the present disclosure, the heat-assisted digestion couldbe completed in minutes and thus it is compatible in both dispersive andan online column format. For reactions in the vials, with a 200 μLvolume of reaction, the ramp up time can be problematic since it takesminutes for the mixture to reach the set temperature. Depending on thetube and thermos-mixer that are used, the ramp up time can vary. Inorder to achieve high reproducibility for this process, robust heattransfer efficiency is important. Commercially available PCR tubesmostly made from polypropylene have shown varied wall thickness. FIG. 15is a graph displaying digestion efficiency comparison among examples.Specifically, FIG. 15 is a graph displaying digestion efficiencycomparison among Smart Digest™ and immobilized prototypes (i.e.,silica-based solid supports with coating of the present technology) inPCR tubes by 5 different vendors. FIG. 16 displays a typical reactionset-up in a 200 μL PCR tube. Cross sections A, B, and C correspond tothe cross section dimensions of FIG. 17. FIG. 17 displays dimensions ofPCR tubes from the tubes used in FIG. 15. The thickness at the bottom ofthe tube (dimension C) seems to have the least impact on digestion. Forexample, AXYGEN has the thickest A and B dimensions, which possiblycontributed to poor heat transfer that resulted in the worst digestionperformance as shown in FIG. 15.

The variation in wall thickness can affect the digestion performancesignificantly (FIG. 15). Moreover, the material of the tube needs to beconsidered. For example, polypropylene can cause non-specific bindingissues using QuanRecovery Vials and Plates (available from WaterTechnologies Corporation, Milford, Mass.). The non-specific bindingissues can be addressed by introducing plasma treatment. Blockingreagents, even though useful at times, could cause severe noise orpeptide loss for downstream analysis in situations of bovine serumalbumin, surfactants, organic solvents and detergents. 200 μL PCR tubescould be easily transitioned to a PCR plate if more samples need to beprocessed at one time, or even to a 384 well plates.

The present disclosure also includes steps after digestion. For modifiedenzyme, quenching is required to terminate the reaction. The trypsindigestion can be stopped by freezing or by lowering the pH of thereaction below pH 4 by adding formic, acetic, or trifluoroacetic acid.

Immobilized enzyme, if in a dispersive format, can require removal ofthe solid support either by centrifugation, filtration or magnetic beadsremoval. Filter membranes or devices used can be selected to haveminimal non-specific binding of the peptides or other analytes ofinterest.

Automating systems that utilize a plate format for digestion with liquidhandling features, can include denaturation, reduction, alkylation,desalting device, and digestion completed on a heater. If immobilizedenzyme is used, enzymes can be removed through positive pressuremanifold or vacuum-assisted filtration. If modified enzyme in-solutionis used, the reaction can be quenched for downstream analysis.

Automating systems can include a flow through mode of digestion, whereprotein is digested while traveling through a column, a chip or anysurface, with or without pretreatment.

The removal of resin from the reaction mixture can be done in differentways, including centrifugation and filtration. The filtration processmay introduce sample loss since membranes made from cellulose acetate orpolyvinylidene fluoride (PVDF) are known to absorb peptides. Inertmaterials or modified membranes should be considered for maximumrecovery.

In some examples, the loss of sample could be significantly reduced withaddition of low concentrations (˜5-100 mM) of arginine,di-methyl-arginine, guanidine, or other derivatives that presentchaotropic properties to the digestion buffer. These chaotrope reagentsat relatively low concentration could reduce the nonspecific bindingduring filtration while not affecting the enzyme reaction in asignificant way. Alternatively, they could be added during thefiltration step to either pre-treat the filter or simultaneouslyfiltered with the digest to minimize nonspecific binding. These measuresmay ensure decent hydrophobic peptide recovery. For example, FIG. 3A andFIG. 3B illustrate the effect on hydrophobic peptide recovery (FIG. 3A)and digestion efficiency (FIG. 3B) by introducing low concentrations (20mM and 50 mM) additions of arginine. FIG. 3C provides the effect onhydrophobic peptide recovery by introducing 5mM and 20 mM of di-methylarginine. And FIG. 3D provides the effect on digestion efficiency byintroducing 5 mM and 20 mM of di-methyl arginine.

In some examples, the technology provides a method of processing asample including protein. The method can include the following steps:adding the sample to an apparatus containing a buffer and a solidsupport surface comprising a surface coating, wherein the surfacecoating immobilizes enzymes and affinity ligands while reducingundesired interactions between the sample and the solid support surface;immobilizing enzymes on the surface coating for digesting the protein inthe sample; immobilizing affinity ligands on the surface coating fortarget capturing a portion of the sample; digesting the protein in thesample with the immobilized enzymes on the surface coating; targetcapturing the portion of the sample with the immobilized affinityligands on the surface coating; and heating the sample to activatedigestion of the protein.

The step of target capturing the portion of the sample can occur before,during, or after the step of digesting the protein in the sample. Insome embodiments, the step of target capturing a portion of the sampleoccurs before, during, and/or after the step of digesting the protein(e.g., 2 of the 3, or all 3 of the indicated timeframes of digestion).The method can also include tuning a reaction condition to determinewhether the step of target capturing the portion of the sample occursbefore, during, or after the step of digesting the protein in thesample.

The reaction condition can include a buffer composition, reactiontemperature, or reaction pH. The solid support surface can be aparticle. In some examples, the enzymes and the affinity ligands areimmobilized on the same particle. Heating the sample to activatepurification or digestion of the protein in the sample can includedigesting the protein in the sample with the immobilized enzymes at anelevated temperature. There can be more than one buffer when processingthe sample. For example, there can be a target capturing elution bufferand a digesting buffer.

The above method can utilize any type of affinity ligand. Examples ofaffinity ligands that can be utilized include, but are not limited to:immoglobin-binding protein such as protein A, G, L or a mixture thereof.The affinity ligand can also be antigen binding such as an antibody,nanobody, or a mixture thereof. The affinity ligand can also be anaptamers.

The affinity ligand can be immobilized on a solid support with a coatingcovering the solid support surface. The coating can provide a surfacecoverage of at least 5 μmoles/m² and reduce undesired interactionsbetween protein sample and the solid support surface and hasfunctionality to covalently bind the affinity ligand. The solid supporton which the affinity ligand is immobilized can be a nonporous or aporous or magnetic, in the form of a membrane, particle, a monolith, asurface of a device, surface of a microchip. The immobilized affinityligand is packed into a device. The device is a plate well, tip of apipette, a channel on a microchip or a tube with one end or both endfrits.

It will be understood by those skilled in the art that various changesin form and details may be made therein without departing from the scopeof the technology of the present disclosure. For example, alternativemodifications include:

-   -   Format factor of the reaction mixture (50, 100, 200, 500, 1000        μL)    -   Immobilized resin packed in columns, in tips, in filtration        plates    -   Enzymes besides trypsin (chymotrypsin, pepsin, protease K, lysC,        IdeS, Glu-C, Arg-C, Asp N, papain pronase and PNGase F and a        combination)    -   Enzyme modified with different hydrophilic cross-linkers,        modifiers;    -   For some proteins, not every step of the digestion workflow        needs to be performed. Proteins without disulfide bonds do not        need to be reduced and alkylated and oftentimes heating itself        could facilitate the denaturation.

Alternative uses (e.g., for biopharmaceutical applications) include:

-   -   Bioanalysis sample preparation    -   Released glycan sample preparation    -   Subunit analysis sample preparation    -   Proteomics sample preparation

Results EXAMPLE 1 Thermal Stability of Immobilized Enzyme ThroughNanoDSC

NanoDSC is routinely used to study the thermodynamics of proteins orpolymers. In a NanoDSC experiment, the protein sample in a buffersolution was scanned at a certain heat rate (° C./min), during which thepeptide bonds and interactions are disrupted. The unfolding processcontinues until the protein are fully denatured. The temperature at themidpoint of the unfolding process dictates when half the molecules areunfolded, which indicates the thermal stability of the protein. In thisexample we used trypsin to investigate the benefit of immobilization.The NanoDSC run was set at 1° C./min scanning from 10° C. to 100° C. forall samples. Each sample contained ˜1 mg/mL trypsin and ˜330 μL was usedfor was scanned twice, with the second scan serving as the referencescan to account for changes that are induced not by trypsindenaturation. As shown in FIG. 1, when trypsin is immobilized on to asolid support, T_(m) has shifted from ˜45° C. to 80° C. There was alsosignificant improvement by the silica-based prototype on T_(m), with atleast 5° C. increase in comparison with the commercial product SmartDigest™.

EXAMPLE 2 Thermal Stability of Immobilized Trypsin with ModificationThrough NanoDSC

Preferably, two hydrophilic crosslinkers with a preferred length wereselected. As shown in FIG. 2, the T_(m) of crosslinked trypsin hasincreased 5° C. in comparison to unmodified trypsin. Point modificationthat targets a particular amino acid also presented some improvement onT_(m) though not as significant, shown in FIG. 2.

EXAMPLE 3 Nonspecific Binding Test on Surface Chemistry ofImmobilization Support

10 μg of NIST mAb standard digest (Waters) was mixed with 10 μL of resinand diluted to 200 μL with digestion buffer that contains 50 mM Tris,250 mM CaCl₂ and 5% glycerol. The mixture was incubated at 75° C. for 5min on a shaker Eppendorf ThermoMixer® C (available from Eppendorf,Hamburg, Germany) at 1400 rpm and then centrifuged 3,000 g for 1 minbefore 100 μL of supernatant was submitted for LC-MS analysis.

The nonspecific binding effect of two different surface chemistry wasevaluated by mixing a NIST mAb digest with a similar pore size (450˜500Å). After 5 min incubation the peptides are collected for analysis. It'sshown here that among the selected hydrophobic peptides, the modifiedPS-DVB had the worst recovery of all. Similarly, the nonspecific bindingeffect was tested among different hydrophilic coating with the sameamount of trypsin immobilized. Shown in FIG. 4, Smart Digest™ showedworse recovery for all the peptides tested. However, this fundamentaltest reflects only on the nonspecific binding effect after the peptideis digested from the protein.

TABLE 1 Parameters for LC-MS analysis on BioAccord ACQUITY I-Class PLUSDetection: ACUITY TUV Column: ACUITY UPLC BEH C18 column (p/n 186003555)Column temp.: 65° C. Sample temp.: 6° C. Injection volume: 10 μL Flowrate: 0.25 mL/min Mobile phase A: 0.1% formic acid in H2O Mobile phaseB: 0.1% formic acid in acetonitrile Gradient: 1% B over 5 min, 1%-40% Bover 65 min, 15% B over 2 min and 1% B for 14 min ACQUITY RDa DetectorMS system: ACQUITY RDa Detector Ionization mode: ESI positiveAcquisition range: m/z 50-2000 Capillary voltage: 1.2 kV Collisionenergy: 60-120 V Cone voltage: 30 V Desolvation energy: 350° C.Intelligent data capture: on

EXAMPLE 4 Quantification of Released Trypsin

10 μL of resin was diluted to 200 μL with digestion buffer that contains50 mM Tris, 250 mM CaCl₂ and 5% glycerol. The mixture was incubated at75° C. for 30 min and 60 min on a shaker (Eppendorf thermo mixer C) at1400 rpm and then centrifuged 3,000 g for 1 min before 100 μL ofsupernatant was submitted for Fluorescence analysis (Excitation: 280 nm,Emission: 370 nm). Free trypsin dissolved in the digestion buffer with aconcentration ranging from 0.004 mg/mL to 0.4 mg/mL was used forgenerating calibration curve.

The released trypsin was quantified after 30- and 60-min incubation at70° C. Shown in FIG. 5, a ˜20% loss of immobilized trypsin was observedon Smart Digest™ after 30 min incubation while the other prototype(i.e., a silica-based solid support with coating of the presenttechnology) showed less than 10%.

EXAMPLE 5 NIST mAb Digestion with Immobilized Enzyme

Approximately 50 μg of NIST mAb was denatured and reduced in 8 Mguanidine buffer with 5 mM DTT for one hour followed with alkylation for30 min in the dark with 15 mM IAM. The alkylated protein then wasdesalted using NAP-5 columns (GE Healthcare) and mixed with 15 μLimmobilized enzyme. The digestion was conducted at 70° C. for 10 min ona shaker before 100 μL of supernatant was submitted for LC-MS analysis.

Shown in FIG. 6, NIST mAb was used as a model protein to be digested byimmobilized trypsin. The peptide map generated through a standard LC-MSassay exhibited similar overall profiles as in solution digestion,however, Smart Digest™ suffered more severe incomplete digestion, withmore than 20% miss-cleavage while a better prototype showed only ˜8%(FIGS. 7 & 8). Incomplete digestion could be a result of unfavorablesurface chemistry of the immobilization support that induces nonspecificbinding and interaction, and it could also be related to deconjugationof trypsin as discussed in Examples 3 & 4. The tested prototype utilizeda hydrophilic coating that could be tuned in “thickness” to maximizedigestion efficiency. Shown in FIG. 7, best digestion efficiency wasachieved with a preferred coating of the modifier labeled as modifiedprototype 3.

EXAMPLE 6 Effect of Temperature on Digestion Efficiency

Samples were digested at 60, 70, 80° C. according to Example 5 as shownin FIGS. 8A, 8B, and 8C.

EXAMPLE 7 Effect of Ca²⁺ on Digestion Efficiency and Nonspecific Binding

1-50 mM of Ca²⁺ was evaluated for its impact on digestion according toExample 5 as shown in FIGS. 9A and 9B.

EXAMPLE 8 Effect of pH on Digestion Efficiency and Deamidation

Samples were digested at pH 7.6 and 6.6 according to Example 5, shownbelow in Table 2.

TABLE 2 pH of buffer measured at different temperature the pH of 50 mMthe pH for Temperature Tris solution immobilized trypsin 25° C. 7.6 6.670° C. 7.0 5.8

EXAMPLE 9 Effect of Additives on Digestion Efficiency

5% of polyols were added to digestion buffer and samples were digestionaccording to Example 5. 50 mM methionine was evaluated for its effect onpreventing artificial oxidation. 5%, 10% acetonitrile were added todigestion buffer to evaluate its effect on reducing artificialdeamidation as shown in FIG. 14.

EXAMPLE 10 PCR Tubes Screening

Samples were digested in 5 different PCR tubes that were from Biologix,Axygen, Andwin Scientific, RPI Scientific and Applied Biosciencesaccording to Example 5 (See FIGS. 15 and 17).

EXAMPLE 11 Tris Concentration Optimization

50, 100, and 200 mM Tris was used as the digestion buffer and proteinsamples were digested at 70° C. according to Example 5 as shown in FIGS.10A and 10B.

EXAMPLE 12 The Effect of Arginine and Dimethyl-Arginine on HydrophobicPeptide Recovery

5, 20 mM of dimethyl-arginine and 20, 50 mM of arginine were added tothe digestion buffer and protein samples were digested at 70° C.according to Example 5 as shown in FIGS. 3A and 3B. All protein digestswere filtered by a PVDF-membrane filter with a positive pressuremanifold.

While this disclosure has been particularly shown and described withreference to example embodiments thereof, it will be understood by thoseskilled in the art that various changes in form and details may be madetherein without departing from the scope of the technology encompassedby the appended claims. For example, other chromatography systems ordetection systems can be used.

1. A method of digesting a sample comprising protein, the method comprising: adding the sample to an apparatus containing a buffer and a solid support surface comprising a surface coating, wherein the surface coating immobilizes enzyme while reducing undesired interactions between the sample and the solid support surface; immobilizing enzymes on the surface coating for digestion of the protein of the sample; and heating the sample to complete a heat-assisted digestion of the protein.
 2. The method of claim 1, wherein the buffer is in-solution.
 3. The method of claim 2, wherein the buffer is selected from the group consisting of: Tris, BIS-Tris, MES, HEPES, triethanolamine, and triethylamine.
 4. The method of claim 2, wherein pH of solution within the apparatus is between 5.0 and 9.0.
 5. The method of claim 2, wherein the buffer in-solution further comprises one or more additives selected from the group consisting of xylitol, methionine, and CaCl₂.
 6. The method of claim 1, wherein complete digestion includes digestion of the protein to less than 15% missed cleavage.
 7. The method of claim 1, wherein the protein sample is heated and digested with immobilized enzyme and complete digestion occurs within 10 minutes.
 8. The method of claim 1, wherein heating the sample to complete digestion of the protein occurs in under 5 minutes with immobilized enzymes.
 9. The method of claim 1, wherein heating occurs at an elevated temperature ranging above 45° C. preferably 65° C. to 75° C. and not greater than 85° C.
 10. The method of claim 9, wherein heating is applied using a device selected from a group consisting of: oven, incubator, rocker, thermomixer.
 11. The method of claim 1, wherein the surface coating comprises two portions, a first coating portion with functionality for bioconjugation and a second coating portion with a functionality to reduce the undesired interactions between the protein and the solid support surface.
 12. The method of claim 11, wherein the surface coating after immobilization comprises a hydrophilic coating with immobilized enzymes attached to its surface.
 13. The method of claim 11, wherein the surface coating provides a surface coverage of at least 5 μmoles/m² on a surface of the solid support.
 14. The method of claim 1, wherein immobilized enzymes are chosen from a group consisting trypsin, chymotrypsin, Lys-C, pepsin Glu-C, Arg-C, Asp N. papain, elastase, IdeS, IdeZ, PNGase F or any combination.
 15. The method of claim 1, wherein the enzyme is modified with hydrophilic crosslinking groups to increase chemical and thermal stability.
 16. The method of claim 1, wherein the enzymes are modified with hydrophobic modifiers to increase affinity to the protein sample.
 17. The method of claim 1, wherein the solid support comprises a polymer-based material, silica based material, hybrid material, agarose, or cellulose.
 18. The method of claim 1, wherein the solid support on which the enzyme is immobilized is a nonporous or a porous or magnetic, in the form of a membrane, particle, a monolith, a surface of a device, surface of a microchip.
 19. The method of claim 1, wherein an overall process of digesting the protein of the sample includes one or more pretreatment steps of the sample prior to heating the sample to complete digestion.
 20. The method of claim 19, wherein the one or more pretreatment steps comprises denaturing the protein of the sample, reducing the protein sample, alkylating the protein sample, or desalting the protein of the sample, or any combinations.
 21. The method of claim 1, wherein heating the sample to complete digestion provides digesting the protein with less than about 15% missed cleavages and greater than about 85% sequence coverage.
 22. The method of claim 1, further comprising submitting the digested protein to downstream analysis comprising at least one of liquid chromatography-ultraviolet (LC-UV) or liquid chromatography-mass spectrometry (LC-MS) 23-43. (canceled) 